The long-term goal is to understand intracellular mechanisms responsible for the control of active transport of chloride ions by the intestinal epithelium. This transport mechanism is the major driving force for the movement of water into the intestinal lumen. Accordingly, significant disease can result from both its under- and over-expression. Studies accomplished during the current funding period have firmly established that there are both positive and negative signalling pathways that interact to set the overall level of chloride secretion. Certain calcium- dependent agonists, such as carbachol, have dual effects on secretion, first stimulating then inhibiting subsequent chloride secretion. Peptide growth factors, such as epidermal growth factor (EGF), have been identified as epithelial agonists that inhibit chloride secretion without stimulating the process. Although carbachol and EGF inhibit chloride secretion via separate pathways, the pathways nevertheless interact because both agents activate the EGF receptor in mediating their inhibitory effects. The goal of the current studies is to define the precise mechanisms underlying the inhibitory effects of carbachol and EGF. Three specific aims are proposed. The first aim will focus on inhibition by carbachol, and define the upstream signals that lead to EGF receptor phosphorylation as well as the downstream targets of carbachol-stimulated mitogen-activated protein kinase activation. The second aim will test the hypothesis that specific isoform(s) of protein kinase C mediate the inhibitory effects of EGF on chloride secretion. The third aim will define the molecular basis of inhibitory signalling via the EGF receptor activated in response to carbachol ("transactivation") versus EGF binding. The overall strategy will be to correlate biochemical and electrophysiological parameters in a well-characterized model human colonic epithelium, the T/84 cell line. The significance of he studies lies in the potential to increase our understanding of the cellular abnormalities in secretory diarrhea, cystic fibrosis and related disorders. Ultimately, improved knowledge of such abnormalities may improve therapies for these conditions.